We thank members of the Mariani lab for critical reading of the manuscript. Our funding sources were: the Baxter Medical Scholar Research Fellowship (to M.K.S.), USC undergraduate fellowships and the Provost, Dean Joan M. Schaeffer, and Rose Hills Fellowships, (to M.K.S.). We also acknowledge a CIRM BRIDGES fellowship through Pasadena City College (to T.T.T). and the James H. Zumberge Research and Innovation Fund, the USC Regenerative Medicine Initiative, and the NIAMS NIH under Award Number R21AR064462 (to F.V.M).

NOTA: Todos os procedimentos estão em conformidade com um protocolo de animais aprovado pelo cuidado e uso Comitê de animal Institucional (IACUC) da Universidade do Sul da Califórnia. 1. Preparação para a cirurgia <ol><li> Separe as ferramentas e suprimentos cirúrgicos necessários (esterilizado, itens 1-18 na lista de materiais) e organizar um microscópio de dissecação e uma fibra óptica trans-iluminador em uma capa ou em uma mesa de corrente descendente. </li><li> Coloque um rato sexualmente maduros (20-30 g, 1-3 meses de idade) em uma câmara de indução com 4% de isoflurano para induzir a anestesia. </li><li> Confirme a anestesia com uma pata e / ou teste de cauda pitada. </li><li> Aplicar pomada para os olhos do rato para evitar o ressecamento do olho durante a cirurgia. </li><li> Mova o rato para o estágio do microscópio. Posicione o mouse para dentro do cone do nariz da mangueira de manutenção e posicionar lateralmente. Proporcionar uma bolsa de aquecimento adjacente ao animal para ajudar a manter a temperatura corporal. NÃOE:. Posição do rato é dependente de preferência pessoal e / ou destreza manual do cirurgião, do lado esquerdo ou direito está bem. </li><li> Ajuste isoflurano a 2 - 3% para manutenção, e assistir a freqüência respiratória. </li><li> Injectar buprenorfina (0,05 mg / kg) subcutaneamente (flanco lateral da perna) para a dor pós-operatória com uma agulha 25 G. A perna pode contorcer-se que é uma resposta normal. </li></ol> Incision 2. Abertura <ol><li> Detectar a costela desejado por palpação, e limpar a área de cabelo com uma máquina de barbear. NOTA: Reforços 8-10 são recomendados desde costelas 1-7 estão mais próximos dos pulmões e 11-13 são consideravelmente mais curto. Contando as costelas deve começar com a costela mais rostral como número 1. </li><li> Preparar a área alternando com povidona-iodo e de isopropanol (70%) raspagem (3x é frequentemente recomendada pelos protocolos de utilização de animais). </li><li> Corte de 2 cm da incisão através da pele, directamente por cima e paralela à nervura desejado com uma tesoura de microcirurgia e média dimensão. IncÉe através das camadas musculares e adiposas subjacentes. </li><li> Coloque todos os três camadas (pele, músculo, gordura) em um retractor para expor o local cirúrgico, minimizando o tamanho da incisão. </li></ol> 3. excisão do Rib <ol><li> Corte através dos músculos intercostais que recobrem a seção desejada da costela com um bisturi 5,0 milímetros. Posição esta incisão de cerca de 5 mm proximal à articulação do condrocostais como este é onde a costela não é tão altamente curvado. Separe cuidadosamente o músculo do osso com uma pinça de ponta fina. </li><li> Para criar uma ressecção, mantendo o periósteo no animal, por meio de corte do periósteo, ao longo do comprimento da nervura com um bisturi 5,0 milímetros. Separe cuidadosamente o periósteo do osso subjacente lateralmente com uma pinça de ponta fina. Proceda com cuidado, como o periósteo é muito delicada e tem uma consistência gelatinosa. </li><li> Em seguida, faça um corte transversal através do osso numa extremidade com micro-tesouras finas. Se necessário, medir a ressecção comum retículo no microscópio ou um medidor tipo régua. Em seguida, levantar cuidadosamente o osso para fora do periósteo e cortar na outra extremidade. NOTA: Proceda com cuidado. Esta é a etapa mais delicada, como puxar para cima sem excisão do costela abaixo vai rasgar a membrana pleural e resultar em um pneumotórax. Se a membrana pleural é rasgada este será facilmente evidente como o interior da cavidade torácica, tornam-se visíveis. </li><li> Se o sangramento ocorre como um resultado de corte do osso, aplicar pressão na extremidade de corte com um cotonete para 4-5 segundos para parar a hemorragia. NOTA: Nas raras ocasiões em que o sangramento continua, pode ser necessário parar e sacrificar o animal a perda de sangue tão significativa comprometerá recuperação (sem reposição de líquidos, o volume máximo de sangue que pode ser seguramente ser perdido é de 10% do volume total de sangue ou 7.7 -. 8 mL / g para 25 g um rato, isso é equivalente a cerca de 180-200 mL 9). </li><li> Colocar imediatamente a nervura removido emPFA 4% para análise futura. NOTA: Se a ressecção de ambos os ossos e do periósteo é para ser realizada, omitir o periósteo incisão e separação (passo 3.2). Desde o periósteo é muito bem ligado à membrana pleural proceder com cautela, provocando a costela longe da membrana pleural com uma pinça para evitar rasgos. </li></ol> 4. fechar a incisão <ol><li> Suturar os músculos intercostais ao longo do topo da manga periosteal remanescente com 9-0 suturas (2 suturas são geralmente suficiente). Coloque as suturas diretamente acima das extremidades cortadas do costela, para atuar como indicadores para o local da cirurgia. </li><li> Remover o afastador. Suturar o músculo e gordura sobreposta com 9-0 suturas (3-4 suturas são normalmente suficientes). Em ratinhos com camadas particularmente espessas de músculo e / ou a gordura, suturar cada camada em separado (por exemplo, uma camada de fios de sutura para a camada muscular e uma camada de suturas para a camada de gordura). </li><li> Feche opele com 7-0 (4-5 suturas são normalmente suficientes). </li><li> Fixe a incisão com cola de sutura, apertando as bordas junto com grandes fórceps. </li><li> Lentamente desmamar o mouse para fora isoflurano ajustando primeiro a 1% por alguns minutos e, em seguida, desligue. </li><li> Posicione o mouse sob uma lâmpada de fogo e deixe até que a consciência é recuperada, 5 - 10 min. Depois de consciência é recuperada e durante todo o período de cicatrização, o rato deve mover-se e deambular normalmente e não mostram sinais de angústia. </li></ol> 5. Recuperação e Análise <ol><li> Fornecer o manejo da dor pós-operatória, incluindo a administração de buprenorfina de forma oral de gelatina a 0,5 mg / kg a cada 12 horas para 48 horas. A administração oral não necessita de prender o animal que pode causar dor na região torácica. </li><li> Manter o rato em uma gaiola com livre acesso a comida e água durante o período de cicatrização. Após a incisão é seca, as fêmeas podem ser co-macho alojados enquantos deve permanecer isolada para evitar lutas. </li><li> Após o período de cicatrização, siga o procedimento aprovado para a eutanásia. Retire a caixa torácica para fixação e análise. </li></ol>

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The authors have nothing to disclose

Quando aprendendo este protocolo, determinando onde localizar a incisão inicial pode ser um desafio. No entanto, a prática em ratos sacrificados ajuda o cirurgião saber onde colocar a incisão inicial e expor a costela desejado a ser ressecado. Trabalhando em cadáveres também melhora as habilidades motoras fine-necessárias para remover a porção de costela com ou sem o periósteo. Além disso, alguém novo para este procedimento pode encontrar manipular as ferramentas finas e suturas finas para ser difícil. Enqu...

Debilitante lesão esquelética, artrose crônica, e os graves problemas associados com a cirurgia reconstrutiva impactar a produtividade econômica, o bem-estar da família e qualidade de vida. Enquanto pequenas pausas e lesões podem curar razoavelmente bem, os seres humanos não são capazes de reparar defeitos grandes e, portanto, deve contar com procedimentos reconstrutivos para restaurar a estrutura e função. Reconstrução pode envolver transplante alogênico ou heterogeneic, osso morcellized, andaimes implantados, ou distração osteogênica. Infelizmente, não só existem fatores de morbidade persistentes associados a esses tratamentos, mas a força original do osso reparado é raramente alcançada. Assim, são necessárias novas abordagens clínicas. One-way para desenvolver métodos inovadores para o tratamento de defeitos segmentares é estudar as situações em que o reparo em grande escala ocorre naturalmente. Anfíbios famosa pode regenerar elementos esqueléticos, enquanto os mamíferos são considerados limitados em thé a capacidade. No entanto, desde o início do século 20, alguns relatos de regeneração na costela humana foram publicados sugerindo que os humanos não podem ser tão limitado 1-4. Atualmente este fenômeno é mais conhecido por cirurgiões plásticos que utilizam material de costela para queixo, rosto e orelha de reconstrução, mas não é mais amplamente apreciada 5. Para estudar este reparo em mais detalhes, nós desenvolvemos um modelo cirúrgico usando o mouse. Usando este protocolo, os pesquisadores podem identificar os fatores inatos envolvidos e usar essa informação para facilitar a cicatrização óssea em outras localidades. Há muitas vantagens em usar as costelas como um modelo para o estudo de reparação óssea. Em primeiro lugar, as nervuras circundantes proporcionar um fixador naturais (como comparado a ressecção do fémur 6,7). Isso diminui o risco de morbidade de fixadores internos e externos e simplifica o procedimento cirúrgico. Em segundo lugar, as camadas musculares finas dos wa peitovai fornecer para fácil acesso e excelente visibilidade que fazer o ensaio comparável à conveniência de ressecções calota craniana 8. Em terceiro lugar, em contraste com a calvárias que formam por ossificação intramembranosa, a forma de nervuras ossificação endocondral e crescer em comprimento por meio de extensão em placas de crescimento localizadas em cada extremidade de uma diáfise central. Portanto, a reparação das costelas pode ser mais comparável ao reparo dos ossos longos do esqueleto apendicular. Além disso, verificou-se que em comparação com o fémur, no periósteo da nervura é mais espessa e pode ser mais facilmente manipulado. Assim, os investigadores que desejam para ensaiar a reparação óssea com a finalidade de estudar o periósteo ou testar terapias celulares, agentes farmacológicos, e / ou suportes de tecido podem encontrar este modelo cirúrgico útil. Em resumo, este modelo de ressecção de costela fornece um contexto no qual a estudar reparação óssea em grande escala natural em mamíferos como existe tal modelo em uso geral existe atualmente.

<table border="0" cellpadding="0" cellspacing="0" width="896">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Name of Reagent/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:304px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">Medium sized micro-dissection scissors (Vannas-T&#252;bingen Spring Scissors 5 mm)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Fine micro-dissection scissors (Vannas Spring Scissors - 2mm Cutting Edge)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">15000-04</td>
			<td>curved tip is beneficial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Micro-scalpel 5.0 mm</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>10315-12</td>
			<td>other fine scalpels can be substituted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Dumont 55 forceps</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">11295-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Retractor&#160;</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>17004-05</td>
			<td>adjustability is convenient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Micro-needle holders</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">12060-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">9.0 nylon sutures (Ethilon), taper point best</td>
			<td style="width:160px;">Ethicon</td>
			<td>2819G or similar</td>
			<td>taper point best but reverse cutting is also good</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">7.0 prolene sutures (Prolene)</td>
			<td style="width:160px;">Ethicon</td>
			<td>8700H or similar</td>
			<td>6-0 can be used too, needle point can vary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Large forceps (Adson Forceps)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">11006-12</td>
			<td>other brands are fine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Lubricant Eye Ointment (Akwa Tears)</td>
			<td style="width:160px;">Akorn</td>
			<td style="width:157px;">17478-062-35</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Suture glue (GLUture Topical Tissue Adhesive)</td>
			<td style="width:160px;">Abbot</td>
			<td style="width:157px;">32046-01</td>
			<td>has excellent working time</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Shaver</td>
			<td style="width:160px;">Wahl</td>
			<td>9918-6171 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Clamp lamp</td>
			<td>Zoo Med</td>
			<td style="width:157px;">LF-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Infrared Bulb, 75W</td>
			<td style="width:160px;">Zoo Med</td>
			<td>RS-75</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:275px;">RC2 Rodent Anesthesia System</td>
			<td style="width:160px;">VetEquip&#160;</td>
			<td style="width:157px;">922100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">IsoFlo (Isoflurane)</td>
			<td style="width:160px;">Abbot</td>
			<td style="width:157px;">05260-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Buprenorphine (Buprenex)</td>
			<td style="width:160px;">Reckitt Benckiser</td>
			<td style="width:157px;">12496-0757-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Betadine</td>
			<td style="width:160px;">Purdue Frederick</td>
			<td>67618015017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Flavored Gelatin, raspberry</td>
			<td style="width:160px;">Jell-O</td>
			<td>B000E1FYL0</td>
			<td>made up firm, to the consistency of &#39;jigglers&#39;</td>
		</tr>
	</tbody>
</table>

a surgical procedure for resecting the mouse rib: a model for large-scale long bone repair

Este protocolo introduz investigadores para um novo modelo de grande escala reparação óssea utilizando a costela mouse. O procedimento detalha o seguinte: preparação do animal para a cirurgia, a abertura da parede torácica corpo, expondo a costela desejado a partir dos músculos intercostais circundantes, extirpando a seção desejada da costela sem induzir um pneumotórax, e fechar as incisões. Em comparação com os ossos do esqueleto apendicular, as nervuras são altamente acessível. Além disso, não fixador interno ou externo é necessário uma vez que as nervuras adjacentes proporcionam uma fixação natural. A cirurgia utiliza comercialmente fornecimentos disponíveis, é fácil de aprender, e bem tolerado pelo animal. O procedimento pode ser realizado com ou sem a remoção do periósteo circundante, e, por conseguinte, a contribuição do periósteo ao reparo pode ser avaliada. Os resultados indicam que, se o periósteo é retida, reparação robusta ocorre em 1-2 meses. Esperamos que o uso deste protocoloestimular a pesquisa em reparação de costela e que os resultados irão facilitar o desenvolvimento de novas formas de estimular a reparação óssea em outras localidades ao redor do corpo.

Animais tipicamente recuperar rapidamente a partir deste procedimento, a incisão cura bem, e os animais exibem comportamento normal. O procedimento utiliza fontes comercialmente disponíveis e após a prática, pode ser concluído em menos de 1 hora. Antes de usar este procedimento para gerar os dados experimentais, é importante analisar a ressecção no dia 0 ponto de tempo para determinar se os fragmentos de ossos grandes pode permanecer na zona de ressecção. Uma forma de avaliar o procedimento adequado é avaliar...

Watch this Scientific Journal Video about A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair at JoVE.com

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Um procedimento cirúrgico para ressecção do mouse Rib: Um Modelo de Grande Escala Repair longo osso

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

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Research

JoVE Journal

Medicine

13.0K visualizações.  Keck School of Medicine, University of Southern California. The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Vídeo: A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Uma nova abordagem cirúrgica para administração intratraqueal de agentes bioativos em um modelo de camundongo Fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Medicina

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.

Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application.

Técnica cirúrgica para entrega da Medula Espinhal de Terapias: Demonstração de Processo em Göttingen porquinhos

This is a  compact visual description of a combination of surgical technique and device  for the  delivery of (gene and cell) therapies into the spinal ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Um modelo de pele Rato Fetal de reparação de feridas Scarless

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

A abordagem do rato Round-janela para ototóxicos Delivery Agent: A rápida e técnica confiável para induzir a degeneração das células da cóclea

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

Um Modelo murino Novel de Fístula Arteriovenosa Falha: O procedimento cirúrgico em Detalhe

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Angiogênese cirúrgica em suínos Allotransplantation Tibial: um novo grande osso Animal vascularizado composto Allotransplantation modelo

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

Avaliando o procedimento para realizar cistometria acordada em um modelo de mouse

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

Um procedimento cirúrgico para a administração de drogas para o ouvido interno em um Non-Human primatas Sagui (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

Avaliação histomorfométrica padronizada da osteoartrite em um modelo de rato cirúrgico

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...